A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene
A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene
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DOI：
10.1101/gad.1714608
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发表时间：
2008-11-01
影响因子：
10.5
通讯作者：
Green, Michael R.
中科院分区：
文献类型：
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作者：
Gobeil, Stephane;Zhu, Xiaochun;Green, Michael R.
转移抑制基因抑制转移所需的一个或多个步骤，而不影响原发肿瘤的形成。由于转移过程的复杂性，确定参与转移预防的基因的实验方法的发展一直具有挑战性。在这里，我们描述了一种全基因组RNAi筛选策略，以确定候选转移抑制基因。shrna在弱转移性B16-F0小鼠黑色素瘤细胞中表达后，在三维细胞培养系统中根据增强的卫星集落形成选择，并在小鼠实验转移试验中得到证实。通过这种方法，我们发现了22个基因，它们的敲除增加了转移而不影响原发肿瘤的生长。我们专注于其中一个基因Gas1（生长抑制特异性1），因为我们发现它在高转移性B16-F10黑色素瘤细胞中被大幅下调，这导致了该小鼠细胞系的高转移潜力。我们进一步证明Gas1具有黑色素瘤肿瘤抑制因子的所有预期特性，包括：在自发转移试验中抑制转移，促进细胞传播到继发部位后的细胞凋亡，以及在人类黑色素瘤转移源细胞系和转移性肿瘤样本中频繁下调。因此，我们开发了一种全基因组shRNA筛选策略，可以发现新的转移抑制基因。
Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.